7, 8 and 9-substituted tetracycline compounds

ABSTRACT

7, 8 and 9-substituted tetracycline compounds, methods of treating tetracycline responsive states, and pharmaceutical compositions containing the 7, 8 and 9-substituted tetracycline compounds are described.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/074,466, filed Mar. 4, 2008; which is a continuation of U.S.application Ser. No. 11/454,221, filed Jun. 16, 2006; which is acontinuation of U.S. application Ser. No. 10/866,220, filed Jun. 10,2004; which is a continuation of U.S. application Ser. No. 10/652,713,filed Aug. 28, 2003; which is a continuation of U.S. application Ser.No. 09/894,805, filed Jun. 29, 2001; which claims the benefit of U.S.Provisional Patent Application No. 60/216,656, filed Jul. 7, 2000. Thisapplication is related to International Patent Application Serial No.:PCT/US00/21366, filed Aug. 4, 2000. The entire contents of all theaforementioned patent applications are hereby incorporated herein byreference.

BACKGROUND OF THE INVENTION

The development of the tetracycline antibiotics was the direct result ofa systematic screening of soil specimens collected from many parts ofthe world for evidence of microorganisms capable of producingbactericidal and/or bacteriostatic compositions. The first of thesenovel compounds was introduced in 1948 under the name chlortetracycline.Two years later, oxytetracycline became available. The elucidation ofthe chemical structure of these compounds confirmed their similarity andfurnished the analytical basis for the production of a third member ofthis group in 1952, tetracycline. A new family of tetracyclinecompounds, without the ring-attached methyl group present in earliertetracyclines, was prepared in 1957 and became publicly available in1967; and minocycline was in use by 1972.

Recently, research efforts have focused on developing new tetracyclineantibiotic compositions effective under varying therapeutic conditionsand routes of administration. New tetracycline analogues have also beeninvestigated which may prove to be equal to or more effective than theoriginally introduced tetracycline compounds. Examples include U.S. Pat.Nos. 3,957,980; 3,674,859; 2,980,584; 2,990,331; 3,062,717; 3,557,280;4,018,889; 4,024,272; 4,126,680; 3,454,697; and 3,165,531. These patentsare representative of the range of pharmaceutically active tetracyclineand tetracycline analogue compositions.

Historically, soon after their initial development and introduction, thetetracyclines were found to be highly effective pharmacologicallyagainst rickettsiae; a number of gram-positive and gram-negativebacteria; and the agents responsible for lymphogranuloma venereum,including conjunctivitis, and psittacosis. Hence, tetracyclines becameknown as “broad spectrum” antibiotics. With the subsequent establishmentof their in vitro antimicrobial activity, effectiveness in experimentalinfections, and pharmacological properties, the tetracyclines as a classrapidly became widely used for therapeutic purposes. However, thiswidespread use of tetracyclines for both major and minor illnesses anddiseases led directly to the emergence of resistance to theseantibiotics even among highly susceptible bacterial species bothcommensal and pathogenic (e.g., pneumococci and Salmonella). The rise oftetracycline-resistant organisms has resulted in a general decline inuse of tetracyclines and tetracycline analogue compositions asantibiotics of choice.

SUMMARY OF THE INVENTION

The invention pertains, at least in part, to 7-, 8- or 9-substitutedtetracycline compounds of the formula:

wherein:

-   -   R⁴ and R^(4′) are each methyl;    -   R⁵ is hydrogen or hydroxyl;    -   R⁶ and R^(6′) are each independently hydrogen, methyl, or        hydroxyl;    -   R⁷ is hydrogen, lower alkenyl, lower alkynyl, phenyl,        halophenyl, acyl, heteroaryl, phenylalkynyl, or dimethylamino;        and    -   R⁸ is hydrogen, phenyl, nitrophenyl, halo, or lower alkynyl; and    -   R⁹ is hydrogen, amino, acetamide, or lower alkynyl; and provided        that at least one of R⁷, R⁸, or R⁹ is not hydrogen; and        pharmaceutically acceptable salts thereof.

In an embodiment, the tetracycline compound of the invention is atetracycline derivative wherein R⁵ is hydrogen, R⁶ is methyl, R^(6′) ishydroxyl, and R⁷ is phenyl.

In another embodiment, the tetracycline compound of the invention is adoxycycline derivative, wherein R⁵ is hydroxyl, R⁶ is methyl, and R^(6′)is hydrogen. In a further embodiment, the doxycycline derivativecomprises R⁷ groups such as lower alkenyl (e.g., ethenyl), lower alkynyl(e.g., ethynyl), heteroaryl (e.g., furanyl, dioxenyl, pyrazinyl, orpyridinyl), phenyl, halophenyl or phenylalkynyl. Doxycycline derivativesalso may comprise R⁸ groups such as hydrogen, halogen (e.g., bromine),lower alkynyl (e.g., ethynyl), phenyl, or nitrophenyl. In certainembodiments, R⁷ is ethenyl or ethynyl; and R⁸ and R⁹ are each hydrogen.In another, R⁷ is phenyl, halophenyl or phenylalkynyl; and R⁸ and R⁹ areeach hydrogen. Alternatively, R⁷ is hydrogen, R⁸ is halo, phenyl, ornitrophenyl, and R⁹ is hydrogen. Further, R⁷ may be hydrogen, R⁸ phenyl,and R⁹ amino.

In another embodiment, the tetracycline compound of the invention is ademeclocycline derivative, wherein R⁵ is hydrogen, R⁶ is hydroxyl,R^(6′) is hydrogen, and R⁷ is phenyl. In another embodiment, thetetracycline compound of the invention is aminocycline derivativewherein R⁵ is hydroxyl, R⁶ is hydrogen, R^(6′) is hydrogen, R⁷ isdimethylamino, and R⁹ is lower alkynyl (e.g., ethynyl).

The invention also pertains to a method for treating a tetracyclineresponsive state in a mammal, by administering to a mammal a compound offormula I. In another aspect, the invention relates to the use of acompound of formula Ito treat a tetracycline responsive state. Theinvention also pertains to pharmaceutical compositions comprising acompound of formula I, and to the use of a compound of formula I in themanufacture of a medicament to treat a tetracycline responsive state.

DETAILED DESCRIPTION OF THE INVENTION

The invention pertains to, at least in part, to 7-, 8- or 9-substitutedtetracycline compounds of the formula:

wherein:

-   -   R⁴ and R^(4′) are each methyl;    -   R⁵ is hydrogen or hydroxyl;    -   R⁶ and R^(6′) are each independently hydrogen, methyl, or        hydroxyl;    -   R⁷ is hydrogen, lower alkenyl, lower alkynyl, phenyl,        halophenyl, acyl, phenylalkynyl, heteroaryl, or dimethylamino;        and    -   R⁸ is hydrogen, phenyl, nitrophenyl, halo, or lower alkynyl;

and R⁹ is hydrogen, amino, acetamide, or lower alkynyl; and providedthat at least one of R⁷, R^(8′), or R⁹ is not hydrogen; andpharmaceutically acceptable salts thereof.

The term “tetracycline compound” includes compounds with a similar ringstructure to tetracycline, such as those included in formula I. Someexamples of tetracycline compounds which can be modified include asubstituent at position 7, 8 or 9 include tetracycline, demeclocycline,sancycline, and doxycycline; however, other derivatives and analoguescomprising a similar ring structure are also included. Table 1 depictsthe structure of tetracycline, demeclocycline, sancycline, anddoxycycline.

TABLE I

Tetracycline

Demeclocycline

Doxycycline

Minocycline

The term “7, 8 or 9-substituted tetracycline compounds” includestetracycline compounds with at least one substituent at the 7, 8 and/or9 position, as described in formula I. In an embodiment, the substitutedtetracycline compound is substituted tetracycline derivative (e.g.,wherein R⁴ and R^(4′) are methyl, R⁵ is hydrogen, R⁶ is methyl andR^(6′) is hydroxyl); substituted doxycycline derivative (e.g., whereinR⁴ and R^(4′) are methyl, R⁵ is hydroxyl R⁶ is methyl and R^(6′) ishydrogen); substituted demeclocycline derivative (e.g., R⁵ is hydrogen,R⁶ is to hydroxyl, R^(6′) is hydrogen, R⁷ is chloro and R⁴ and R^(4′)are each methyl); or substituted minocycline derivative (wherein R⁴ andR^(4′) are methyl; R⁵ is hydrogen and R⁶ and R^(6′) are hydrogen atoms).

In an embodiment, the substituted tetracycline compound of the inventionis a tetracycline derivative, wherein R⁵ is hydrogen, R⁶ is methyl,R^(6′) is hydroxyl, and R⁷ is phenyl. Examples of such tetracyclinecompounds include 7-phenyl tetracycline.

In another embodiment, the substituted tetracycline compound of theinvention is a doxycycline derivative, wherein R⁵ is hydroxyl, R⁶ ismethyl, and R^(6′) is hydrogen.

In an alternate embodiment, doxycycline derivatives of tetracyclinecompounds of the invention include compounds wherein R⁷ is hydrogen, R⁸is halo, phenyl, or nitrophenyl, and R⁹ is hydrogen. In anotheralternate embodiment, R⁷ is hydrogen, R⁸ is phenyl or lower alkynyl(e.g., ethynyl), and R⁹ is amino. In yet another alternate embodiment,R⁷ and R⁸ are both hydrogen and R⁹ is acetamide.

In further embodiments of the invention, the doxycycline derivativesinclude compounds wherein R⁷ is lower alkenyl (e.g., ethenyl), loweralkynyl (e.g., ethynyl), phenyl, heteroaryl (e.g., furanyl, pyrazinyl,pyridinyl, or dioxenyl), acyl (e.g., lower alkyl carbonyl, e.g.,methylcarbonyl), halophenyl or phenylalkynyl (e.g., phenylethynyl); andR⁸ and R⁹ are each hydrogen.

Examples of such tetracycline compound which are doxycycline derivativesinclude 7-ethenyl doxycycline, 7-ethynyl doxycycline, 7-phenyldoxycycline, 7-(4-fluorophenyl) doxycycline, 7-phenylethynyldoxycycline, 9-acetamide doxycycline, 8-phenyl doxycycline, 8-bromodoxycycline, 8-(p-nitrophenyl) doxycycline, 8-ethynyl-9-aminodoxycycline, 8-phenyl-9-amino doxycycline, 7-(1,4-dioxenyl) doxycycline,7-(2-furanyl) doxycycline, 7-(2-pyrazinyl) doxycycline, 7-(2-pyridinyl)doxycycline, and 7-acyl doxycycline.

In another embodiment, the tetracycline compound of the invention is ademeclocycline derivative, wherein R⁵ is hydrogen, R⁶ is hydroxyl,R^(6′) is hydrogen, R⁷ is phenyl, and R⁸ and R⁹ are both hydrogen.Examples of demeclocycline derivatives include 7-phenyl demeclocycline.

In another embodiment, the tetracycline compound of the invention isaminocycline derivative, wherein R⁵ is hydroxyl, R⁶ is hydrogen, R^(6′)is hydrogen, R⁷ is dimethylamino, R⁸ is hydrogen, and R⁹ is loweralkynyl (e.g., ethynyl). Examples include 9-ethynyl minocycline.

Compounds of the invention can be synthesized by transition metalcatalyzed coupling of tetracyclines halogenated at the 7-, 8-, or9-position. For example, many reactions between aryl halides and variousreactive species have been developed using transition metal catalysis.Coupling of aryl halides or triflates with main group organometallicswith oxidate addition—transmetallation—reductive elimination reactionshas been developed and occurs using a wide range of catalysts, such asPd(Pd₃)₄, Pd(dba)₂, PdCl₂, Pd(OAc)₂, and PdCl₂(CH₃CN)₂ Ligands such asPPh₃ or AsPh₃ may be added to form catalysts in situ with palladiumspecies such as Pd(dba)₂ or PdCl₂. Furthermore, copper salts, such asCuCN or CuI may also be added to further enhance the reaction. Anexample of a coupling using a halogenated tetracycline compound is shownin Scheme 1. In Scheme 1, X is bromine or iodine.

The substituted tetracycline compounds of the invention can besynthesized using organotin reagents, halogenated or triflatetetracycline compounds, and an appropriate catalyst (e.g., palladium).Examples of tin reagents include, for example, ethenyl tributyltin,ethynyl tributyltin, phenyl tributyltin, ethenyl trimethyl tin, ethynyltrimethyl tin, etc. These Stile type couplings are run by adding thetransition metal (e.g., palladium) catalyst to a solution of thehalogenated or triflate tetracycline compound and the organotin reagentin polar solvents. Stille type couplings with alkynyl and alkenyl tinreagents are shown in Scheme 2, wherein X is a halogen or a triflategroup.

Other methods of synthesizing the 7-, 8-, and 9-substituted tetracyclinecompounds of the invention include coupling halogenated tetracyclinecompounds to boronic acids using Suzuki type couplings (M. J. Sharp etal. Tetrahedron Lett. 28 (1987) 5093; W. Cheng, et al. Tetrahedron Lett.28 (1987) 5097; Alves, A. B. et al. Tetrahedron Lett. 29 (1988) 2135; D.Muller, et al. Tetrahedron Lett. 32 (1991) 2135), Grignard reagents (K.Tamao et al. Bull. Chem. Soc. Jpn. 49 (1976) 1958), or organolithiumreagents (S.-I. Murahashi et al., J. Org. Chem 44 (1979) 2408) and atransition metal catalyst, as shown in Scheme 3.

8-halogenated tetracycline compounds can be synthesized viaazidotetracyclines. The protonolysis of the aryl azides produces8-halo-9-amino tetracycline in good yield.

The term “alkyl” includes saturated aliphatic groups, includingstraight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups(isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups(cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkylsubstituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.The term alkyl further includes alkyl groups, which comprise oxygen,nitrogen, sulfur or phosphorous atoms replacing one or more carbons ofthe hydrocarbon backbone. In certain embodiments, a straight chain orbranched chain alkyl has 6 or fewer carbon atoms in its backbone (e.g.,C₁-C₆ for straight chain, C₃-C₆ for branched chain), and more preferably4 or fewer. Likewise, preferred cycloalkyls have from 3-8 carbon atomsin their ring structure, and more preferably have 5 or 6 carbons in thering structure. The term C₁-C₆ includes alkyl groups containing Ito 6carbon atoms.

Moreover, the term alkyl includes both “unsubstituted alkyls” and“substituted alkyls”, the latter of which refers to alkyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, alkyl,alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkylamino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Cycloalkyls can be further substituted, e.g.,with the substituents described above. An “alkylaryl” or an “aralkyl”moiety is an alkyl substituted with an aryl (e.g., phenylmethyl(benzyl)). The term “alkyl” also includes the side chains of natural andunnatural amino acids.

The term “aryl” includes groups with aromaticity, including 5- and6-membered single-ring aromatic groups that may include from zero tofour heteroatoms as well as multicyclic systems with at least onearomatic ring. Examples of aryl groups include benzene, phenyl, pyrrole,furan, thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole,pyrazole, oxazole, isooxazole, pyridine, pyrazine, pyridazine, andpyrimidine, and the like. Furthermore, the term “aryl” includesmulticyclic aryl groups, e.g., tricyclic, bicyclic, e.g., naphthalene,benzoxazole, benzodioxazole, benzothiazole, benzoimidazole,benzothiophene, methylenedioxyphenyl, quinoline, isoquinoline,napthridine, indole, benzofuran, purine, benzofuran, deazapurine, orindolizine. Those aryl groups having heteroatoms in the ring structuremay also be referred to as “aryl heterocycles”, “heterocycles,”“heteroaryle” or “heteroaromatics”. The aromatic ring can be substitutedat one or more ring positions with such substituents as described above,as for example, halogen, hydroxyl, alkoxy, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, alkylaminocarbonyl, aralkylaminocarbonyl,alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl,alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Aryl groups can also be fused or bridged withalicyclic or heterocyclic rings which are not aromatic so as to form amulticyclic system (e.g., tetralin, methylenedioxyphenyl). In a furtherembodiment, the term “heteroaryl” refers to pyrazinyl, pyridinyl,furanyl, and dioxenyl groups.

The term “alkenyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, but thatcontain at least one double bond.

For example, the term “alkenyl” includes straight-chain alkenyl groups(e.g., ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl,nonenyl, decenyl, etc.), branched-chain alkenyl groups, cycloalkenyl(alicyclic) groups (cyclopropenyl, cyclopentenyl, cyclohexenyl,cycloheptenyl, cyclooctenyl), alkyl or alkenyl substituted cycloalkenylgroups, and cycloalkyl or cycloalkenyl substituted alkenyl groups. Theterm alkenyl further includes alkenyl groups which include oxygen,nitrogen, sulfur or phosphorous atoms replacing one or more carbons ofthe hydrocarbon backbone. In certain embodiments, a straight chain orbranched chain alkenyl group has 6 or fewer carbon atoms in its backbone(e.g., C₂-C₆ for straight chain, C₃-C₆ for branched chain). Likewise,cycloalkenyl groups may have from 3-8 carbon atoms in their ringstructure, and more preferably have 5 or 6 carbons in the ringstructure. The term C₂-C₆ includes alkenyl groups containing 2 to 6carbon atoms.

Moreover, the term alkenyl includes both “unsubstituted alkenyls” and“substituted alkenyls”, the latter of which refers to alkenyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, alkylgroups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, allylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. In a further embodiment, the term “loweralkenyl” refers to moieties with 2-5 carbons. Advantageous lower alkenylgroups include ethenyl groups.

The term “alkynyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, butwhich contain at least one triple bond.

For example, the term “alkynyl” includes straight-chain alkynyl groups(e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl,nonynyl, decynyl, etc.), branched-chain alkynyl groups, and cycloalkylor cycloalkenyl substituted alkynyl groups. The term alkynyl furtherincludes alkynyl groups which include oxygen, nitrogen, sulfur orphosphorous atoms replacing one or more carbons of the hydrocarbonbackbone. In certain embodiments, a straight chain or branched chainalkynyl group has 6 or fewer carbon atoms in its backbone (e.g., C₂-C₆for straight chain, C₃-C₆ for branched chain). The term C₂-C₆ includesalkynyl groups containing 2 to 6 carbon atoms.

Moreover, the term alkynyl includes both “unsubstituted alkynyls” and“substituted alkynyls”, the latter of which refers to alkynyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, alkylgroups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto five carbon atoms in its backbone structure, e.g., methyl, ethyl,propyl, butyl and pentyl. “Lower alkenyl” and “lower alkynyl” have chainlengths of, for example, 2-5 carbon atoms, e.g., ethenyl, ethynyl,propenyl, propynyl, butenyl, butynyl, pentenyl, and pentynyl.

The term “acyl” includes compounds and moieties which contain the acylradical (CH₃CO—) or a carbonyl group. The term “substituted acyl”includes acyl groups where one or more of the hydrogen atoms arereplaced by for example, alkyl groups, alkynyl groups, halogens,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano,amino (including alkylamino, dialkylamino, arylamino, diarylamino, andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

The term “acylamino” includes moieties wherein an acyl moiety is bondedto an amino group. For example, the term includes alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido groups.

The term “aroyl” includes compounds and moieties with an aryl orheteroaromatic moiety bound to a carbonyl group. Examples of aroylgroups include phenylcarboxy, naphthyl carboxy, etc.

The terms “alkoxyalkyl”, “alkylaminoalkyl” and “thioalkoxyalkyl” includealkyl groups, as described above, which further include oxygen, nitrogenor sulfur atoms replacing one or more carbons of the hydrocarbonbackbone, e.g., oxygen, nitrogen or sulfur atoms. The term “alkoxy”includes substituted and unsubstituted alkyl, alkenyl, and alkynylgroups covalently linked to an oxygen atom. Examples of alkoxy groupsinclude methoxy, ethoxy, isopropyloxy, propoxy, butoxy, and pentoxygroups. Examples of substituted alkoxy groups include halogenated alkoxygroups. The alkoxy groups can be substituted with groups such asalkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkylamino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moieties. Examples of halogen substituted alkoxygroups include, but are not limited to, fluoromethoxy, difluoromethoxy,trifluoromethoxy, chloromethoxy, dichloromethoxy, trichloromethoxy, etc.

The term “amide” or “aminocarboxy” includes compounds or moieties whichcontain a nitrogen atom which is bound to the carbon of a carbonyl or athiocarbonyl group. The term includes “alkaminocarboxy” groups whichinclude alkyl, alkenyl, or alkynyl groups bound to an amino group boundto a carboxy group. It includes arylaminocarboxy groups which includearyl or heteroaryl moieties bound to an amino group which is bound tothe carbon of a carbonyl or thiocarbonyl group. The terms“alkylaminocarboxy,” “alkenylaminocarboxy,” “alkynylaminocarboxy,” and“arylaminocarboxy” include moieties wherein alkyl, alkenyl, alkynyl andaryl moieties, respectively, are bound to a nitrogen atom which is inturn bound to the carbon of a carbonyl group.

The term “amine” or “amino” includes compounds where a nitrogen atom iscovalently bonded to at least one carbon or heteroatom. The term“alkylamino” includes groups and compounds wherein the nitrogen is boundto at least one additional alkyl group. The term “dialkylamino” includesgroups wherein the nitrogen atom is bound to at least two additionalalkyl groups. The term “arylamino” and “diarylamino” include groupswherein the nitrogen is bound to at least one or two aryl groups,respectively. The term “alkylarylamino,” “alkylaminoaryl” or“arylaminoalkyl” refers to an amino group which is bound to at least onealkyl group and at least one aryl group. The term “alkaminoalkyl” refersto an alkyl, alkenyl, or alkynyl group bound to a nitrogen atom which isalso bound to an alkyl group.

The term “carbonyl” or “carboxy” includes compounds and moieties whichcontain a carbon connected with a double bond to an oxygen atom.Examples of moieties which contain a carbonyl include aldehydes,ketones, carboxylic acids, amides, esters, anhydrides, etc.

The term “dioxenyl” refers to moieties of the formula:

The term “ester” includes compounds and moieties which contain a carbonor a heteroatom bound to an oxygen atom which is bonded to the carbon ofa carbonyl group. The term “ester” includes alkoxycarboxy groups such asmethoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl,pentoxycarbonyl, etc. The alkyl, alkenyl, or alkynyl groups are asdefined above.

The term “ether” includes compounds or moieties which contain an oxygenbonded to two different carbon atoms or heteroatoms. For example, theterm includes “alkoxyalkyl” which refers to an alkyl, alkenyl, oralkynyl group covalently bonded to an oxygen atom which is covalentlybonded to another alkyl group.

The term “halo” includes, for example, substituents such as chlorine,fluorine, bromine, or iodine, as well as mono-, di- or tri-halgentatedlower alkyl group, e.g., mono-, di- or tri-halogenated methyl groups. Incertain embodiments, the halo substitution of the phenyl substituentenhances the ability of the tetracycline compound to perform itsintended function, e.g., treat tetracycline responsive states.

The term “heteroatom” includes atoms of any element other than carbon orhydrogen. Examples of heteroatoms include nitrogen, oxygen, sulfur andphosphorus.

The term “hydroxy” or “hydroxyl” includes groups with an —OH or —O⁻.

The terms “polycyclyl” or “polycyclic radical” refer to two or morecyclic rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, arylsand/or heterocyclyls) in which two or more carbons are common to twoadjoining rings. Rings that are joined through non-adjacent atoms aretermed “bridged” rings. Each of the rings of the polycycle can besubstituted with such substituents as described above, as for example,halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl,alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl,aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato,phosphinato, cyano, amino (including alkylamino, dialkylamino,arylamino, diarylamino, and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkyl, alkylaryl, or anaromatic or heteroaromatic moiety.

The term “thiocarbonyl” or “thiocarboxy” includes compounds and moietieswhich contain a carbon connected with a double bond to a sulfur atom.

The term “thioether” includes compounds and moieties which contain asulfur atom bonded to two different carbon or hetero atoms. Examples ofthioethers include, but are not limited to alkthioalkyls,alkthioalkenyls, and alkthioalkynyls. The term “alkthioalkyls” includecompounds with an alkyl, alkenyl, or alkynyl group bonded to a sulfuratom which is bonded to an alkyl group. Similarly, the term“alkthioalkenyls” and alkthioalkynyls” refer to compounds or moietieswherein an alkyl, alkenyl, or alkynyl group is bonded to a sulfur atomwhich is covalently bonded to an alkynyl group.

It will be noted that the structure of some of the compounds of thisinvention includes asymmetric carbon atoms. It is to be understoodaccordingly that the isomers arising from such asymmetry (e.g., allenantiomers and diastereomers) are included within the scope of thisinvention, unless indicated otherwise. Such isomers can be obtained insubstantially pure form by classical separation techniques and bystereochemically controlled synthesis. Furthermore, the structures andother compounds and moieties discussed in this application also includeall tautomers thereof.

Prodrugs are compounds which are converted in vivo to active forms (see,e.g., R. B. Silverman, 1992, “The Organic Chemistry of Drug Design andDrug Action”, Academic Press, Chp. 8). Prodrugs can be used to alter thebiodistribution (e.g., to allow compounds which would not typicallyenter the reactive site of the protease) or the pharmacokinetics for aparticular compound. For example, a hydroxyl group, can be esterified,e.g., with a carboxylic acid group to yield an ester. When the ester isadministered to a subject, the ester is cleaved, enzymatically ornon-enzymatically, reductively or hydrolytically, to reveal the hydroxylgroup.

The term “prodrug moiety” includes moieties which can be metabolized invivo to a hydroxyl group and moieties which may advantageously remainesterified in vivo. Preferably, the prodrugs moieties are metabolized invivo by esterases or by other mechanisms to hydroxyl groups or otheradvantageous groups. Examples of prodrugs and their uses are well knownin the art (See, e.g., Berge et al. (1977) “Pharmaceutical Salts”, J.Pharm. Sci. 66:1-19). The prodrugs can be prepared in situ during thefinal isolation and purification of the compounds, or by separatelyreacting the purified compound in its free acid form or hydroxyl with asuitable esterifying agent. Hydroxyl groups can be converted into estersvia treatment with a carboxylic acid. Examples of prodrug moietiesinclude substituted and unsubstituted, branch or unbranched lower alkylester moieties, (e.g., propionoic acid esters), lower alkenyl esters,di-lower alkyl-amino lower-alkyl esters (e.g., dimethylaminoethylester), acylamino lower alkyl esters (e.g., acetyloxymethyl ester),acyloxy lower alkyl esters (e.g., pivaloyloxymethyl ester), aryl esters(phenyl ester), aryl-lower alkyl esters (e.g., benzyl ester),substituted (e.g., with methyl, halo, or methoxy substituents) aryl andaryl-lower alkyl esters, amides, lower-alkyl amides, di-lower alkylamides, and hydroxy amides. Preferred prodrug moieties are propionoicacid esters and acyl esters.

The invention also features a method for treating a tetracyclinecompound responsive state in a subject, by administering to the subjecta 7-, 8-, or 9-substituted tetracycline compound of the invention, e.g.,a compound of formula I. Preferably, an effective amount of thetetracycline compound is administered. Examples of 7, 8 or 9-substitutedtetracycline compounds of the invention include 7-phenyl tetracycline,7-ethenyl doxycycline 7-ethynyl doxycycline, 7-phenyl doxycycline,7-(4-fluorophenyl) doxycycline, 7-phenylethynyl doxycycline, 9-acetamidedoxycycline, 8-phenyl doxycycline, 8-bromo doxycycline,8-(p-nitrophenyl) doxycycline, 8-ethynyl-9-amino doxycycline,8-phenyl-9-amino doxycycline, 7-phenyl demeclocycline, and 9-ethynylminocycline, 7-(1,4-dioxenyl) doxycycline, 7-(2-furanyl) doxycycline,7-(2-pyrazinyl) doxycycline, 7-(2-pyridinyl) doxycycline, and 7-acetyldoxycycline.

The language “tetracycline compound responsive state” includes stateswhich can be treated, prevented, or otherwise ameliorated by theadministration of a tetracycline compound of the invention. Tetracyclinecompound responsive states include bacterial infections (including thosewhich are resistant to other tetracycline compounds), cancer, diabetes,and other states for which tetracycline compounds have been found to beactive (see, for example, U.S. Pat. Nos. 5,789,395; 5,834,450; and5,532,227, incorporated herein by reference). Compounds of the inventioncan be used to prevent or control important mammalian and veterinarydiseases such as diarrhea, urinary tract infections, infections of skinand skin structure, ear, nose and throat infections, wound infection,mastitis and the like. In addition, methods for treating neoplasms usingtetracycline compounds of the invention are also included (van derBozert et al., Cancer Res., 48:6686-6690 (1988)).

Bacterial infections may be caused by a wide variety of gram positiveand gram negative bacteria. The compounds of the invention are useful asantibiotics against organisms which are resistant to other tetracyclinecompounds. The antibiotic activity of the tetracycline compounds of theinvention may be determined using the method discussed in Example 2, orby using the in vitro standard broth dilution method described in Waitz,J. A., National Commission for Clinical Laboratory Standards, DocumentM7-A2, vol. 10, no. 8, pp. 13-20, 2^(nd) edition, Villanova, Pa. (1990).

The tetracycline compounds may also be used to treat infectionstraditionally treated with tetracycline compounds such as, for example,rickettsiae; a number of gram-positive and gram-negative bacteria; andthe agents responsible for lymphogranuloma venereum, inclusionconjunctivitis, psittacosis. The tetracycline compounds may be used totreat infections of, e.g., K. pneumoniae, Salmonella, E. hirae, A.baumanii, B. catarrhalis, H. influenzae, P. aeruginosa, E. faecium, E.coli, S. aureus or E. faecalis. In one embodiment, the tetracyclinecompound is used to treat a bacterial infection that is resistant toother tetracycline antibiotic compounds. The tetracycline compound ofthe invention may be administered with a pharmaceutically acceptablecarrier.

The language “effective amount” of the compound is that amount necessaryor sufficient to treat or prevent a tetracycline compound responsivestate. The effective amount can vary depending on such factors as thesize and weight of the subject, the type of illness, or the particulartetracycline compound. For example, the choice of the tetracyclinecompound can affect what constitutes an “effective amount”. One ofordinary skill in the art would be able to study the aforementionedfactors and make the determination regarding the effective amount of thetetracycline compound without undue experimentation.

The invention also pertains to methods of treatment againstmicroorganism infections and associated diseases. The methods includeadministration of an effective amount of one or more tetracyclinecompounds to a subject. The subject can be either a plant or,advantageously, an animal, e.g., a mammal, e.g., a human.

In the therapeutic methods of the invention, one or more tetracyclinecompounds of the invention may be administered alone to a subject, ormore typically a compound of the invention will be administered as partof a pharmaceutical composition in mixture with conventional excipient,i.e., pharmaceutically acceptable organic or inorganic carriersubstances suitable for parenteral, oral or other desired administrationand which do not deleteriously react with the active compounds and arenot deleterious to the recipient thereof.

In one embodiment, the pharmaceutical composition comprises a 7-, 8 or9-substituted tetracycline compound of the invention, e.g., of formulaI. In a further embodiment, the 7, 8 or 9-substituted tetracyclinecompound is 7-phenyl tetracycline, 7-ethenyl doxycycline 7-ethynyldoxycycline, 7-phenyl doxycycline, 7-(4-fluorophenyl) doxycycline,7-phenylethynyl doxycycline, 9-acetamide doxycycline, 8-phenyldoxycycline, 8-bromo doxycycline, 8-(p-nitrophenyl) doxycycline,8-ethynyl-9-amino doxycycline, 8-phenyl-9-amino doxycycline, 7-phenyldemeclocycline, and 9-ethynyl minocycline, 7-(1,4-dioxenyl) doxycycline,7-(2-furanyl) doxycycline, 7-(2-pyrazinyl) doxycycline, 7-(2-pyridinyl)doxycycline, or 7-acetyl doxycycline.

The language “pharmaceutically acceptable carrier” includes substancescapable of being coadministered with the tetracycline compound(s), andwhich allow both to perform their intended function, e.g., treat orprevent a tetracycline compound responsive state. Suitablepharmaceutically acceptable carriers include but are not limited towater, salt solutions, alcohol, vegetable oils, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, perfume oil, fatty acid monoglycerides anddiglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose,polyvinylpyrrolidone, etc. The pharmaceutical preparations can besterilized and if desired mixed with auxiliary agents, e.g., lubricants,preservatives, stabilizers, wetting agents, emulsifiers, salts forinfluencing osmotic pressure, buffers, colorings, flavorings and/oraromatic substances and the like which do not deleteriously react withthe active compounds of the invention.

The tetracycline compounds of the invention that are basic in nature arecapable of forming a wide variety of salts with various inorganic andorganic acids. The acids that may be used to prepare pharmaceuticallyacceptable acid addition salts of the tetracycline compounds of theinvention that are basic in nature are those that form non-toxic acidaddition salts, i.e., salts containing pharmaceutically acceptableanions, such as the hydrochloride, hydrobromide, hydroiodide, nitrate,sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate,lactate, salicylate, citrate, acid citrate, tartrate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucaronate, saccharate, formate, benzoate, glutamate,methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonateand palmoate [i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)] salts.Although such salts must be pharmaceutically acceptable foradministration to a subject, e.g., a mammal, it is often desirable inpractice to initially isolate a tetracycline compound of the inventionfrom the reaction mixture as a pharmaceutically unacceptable salt andthen simply convert the latter back to the free base compound bytreatment with an alkaline reagent and subsequently convert the latterfree base to a pharmaceutically acceptable acid addition salt. The acidaddition salts of the base compounds of this invention are readilyprepared by treating the base compound with a substantially equivalentamount of the chosen mineral or organic acid in an aqueous solventmedium or in a suitable organic solvent, such as methanol or ethanol.Upon careful evaporation of the solvent, the desired solid salt isreadily obtained. The preparation of other tetracycline compounds of theinvention not specifically described in the foregoing experimentalsection can be accomplished using combinations of the reactionsdescribed above that will be apparent to those skilled in the art.

The preparation of other tetracycline compounds of the invention notspecifically described in the foregoing experimental section can beaccomplished using combinations of the reactions described above thatwill be apparent to those skilled in the art.

The tetracycline compounds of the invention that are acidic in natureare capable of forming a wide variety of base salts. The chemical basesthat may be used as reagents to prepare pharmaceutically acceptable basesalts of those tetracycline compounds of the invention that are acidicin nature are those that form non-toxic base salts with such compounds.Such non-toxic base salts include, but are not limited to those derivedfrom such pharmaceutically acceptable cations such as alkali metalcations (e.g., potassium and sodium) and alkaline earth metal cations(e.g., calcium and magnesium), ammonium or water-soluble amine additionsalts such as N-methylglucamine-(meglumine), and the loweralkanolammonium and other base salts of pharmaceutically acceptableorganic amines. The pharmaceutically acceptable base addition salts oftetracycline compounds of the invention that are acidic in nature may beformed with pharmaceutically acceptable cations by conventional methods.Thus, these salts may be readily prepared by treating the tetracyclinecompound of the invention with an aqueous solution of the desiredpharmaceutically acceptable cation and evaporating the resultingsolution to dryness, preferably under reduced pressure. Alternatively, alower alkyl alcohol solution of the tetracycline compound of theinvention may be mixed with an alkoxide of the desired metal and thesolution subsequently evaporated to dryness.

The preparation of other tetracycline compounds of the invention notspecifically described in the foregoing experimental section can beaccomplished using combinations of the reactions described above thatwill be apparent to those skilled in the art.

The tetracycline compounds of the invention and pharmaceuticallyacceptable salts thereof can be administered via either the oral,parenteral or topical routes. In general, these compounds are mostdesirably administered in effective dosages, depending upon the weightand condition of the subject being treated and the particular route ofadministration chosen. Variations may occur depending upon the speciesof the subject being treated and its individual response to saidmedicament, as well as on the type of pharmaceutical formulation chosenand the time period and interval at which such administration is carriedout.

The pharmaceutical compositions of the invention may be administeredalone or in combination with other known compositions for treatingtetracycline responsive states in a mammal. Preferred mammals includepets (e.g., cats, dogs, ferrets, etc.), farm animals (cows, sheep, pigs,horses, goats, etc.), lab animals (rats, mice, monkeys, etc.), andprimates (chimpanzees, humans, gorillas). The language “in combinationwith” a known composition is intended to include simultaneousadministration of the composition of the invention and the knowncomposition, administration of the composition of the invention first,followed by the known composition and administration of the knowncomposition first, followed by the composition of the invention. Any ofthe therapeutically composition known in the art for treatingtetracycline responsive states can be used in the methods of theinvention.

The compounds of the invention may be administered alone or incombination with pharmaceutically acceptable carriers or diluents by anyof the routes previously mentioned, and the administration may becarried out in single or multiple doses. For example, the noveltherapeutic agents of this invention can be administered advantageouslyin a wide variety of different dosage forms, i.e., they may be combinedwith various pharmaceutically acceptable inert carriers in the form oftablets, capsules, lozenges, troches, hard candies, powders, sprays,creams, salves, suppositories, jellies, gels, pastes, lotions,ointments, aqueous suspensions, injectable solutions, elixirs, syrups,and the like. Such carriers include solid diluents or fillers, sterileaqueous media and various non-toxic organic solvents, etc. Moreover,oral pharmaceutical compositions can be suitably sweetened and/orflavored. In general, the therapeutically-effective compounds of thisinvention are present in such dosage forms at concentration levelsranging from about 5.0% to about 70% by weight.

For oral administration, tablets containing various excipients such asmicrocrystalline cellulose, sodium citrate, calcium carbonate, dicalciumphosphate and glycine may be employed along with various disintegrantssuch as starch (and preferably corn, potato or tapioca starch), alginicacid and certain complex silicates, together with granulation binderslike polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally,lubricating agents such as magnesium stearate, sodium lauryl sulfate andtalc are often very useful for tabletting purposes. Solid compositionsof a similar type may also be employed as fillers in gelatin capsules;preferred materials in this connection also include lactose or milksugar as well as high molecular weight polyethylene glycols. Whenaqueous suspensions and/or elixirs are desired for oral administration,the active ingredient may be combined with various sweetening orflavoring agents, coloring matter or dyes, and, if so desired,emulsifying and/or suspending agents as well, together with suchdiluents as water, ethanol, propylene glycol, glycerin and various likecombinations thereof.

For parenteral administration (including intraperitoneal, subcutaneous,intravenous, intradermal or intramuscular injection), solutions of atherapeutic compound of the present invention in either sesame or peanutoil or in aqueous propylene glycol may be employed. The aqueoussolutions should be suitably buffered (preferably pH greater than 8) ifnecessary and the liquid diluent first rendered isotonic. These aqueoussolutions are suitable for intravenous injection purposes. The oilysolutions are suitable for intraarticular, intramuscular andsubcutaneous injection purposes. The preparation of all these solutionsunder sterile conditions is readily accomplished by standardpharmaceutical techniques well known to those skilled in the art. Forparenteral application, examples of suitable preparations includesolutions, preferably oily or aqueous solutions as well as suspensions,emulsions, or implants, including suppositories. Therapeutic compoundsmay be formulated in sterile form in multiple or single dose formatssuch as being dispersed in a fluid carrier such as sterile physiologicalsaline or 5% saline dextrose solutions commonly used with injectables.

Additionally, it is also possible to administer the compounds of thepresent invention topically when treating inflammatory conditions of theskin. Examples of methods of topical administration include transdermal,buccal or sublingual application. For topical applications, therapeuticcompounds can be suitably admixed in a pharmacologically inert topicalcarrier such as a gel, an ointment, a lotion or a cream. Such topicalcarriers include water, glycerol, alcohol, propylene glycol, fattyalcohols, triglycerides, fatty acid esters, or mineral oils. Otherpossible topical carriers are liquid petrolatum, isopropylpalmitate,polyethylene glycol, ethanol to 95%, polyoxyethylene monolauriate 5% inwater, sodium lauryl sulfate 5% in water, and the like. In addition,materials such as anti-oxidants, humectants, viscosity stabilizers andthe like also may be added if desired.

For enteral application, particularly suitable are tablets, dragees orcapsules having talc and/or carbohydrate carrier binder or the like, thecarrier preferably being lactose and/or corn starch and/or potatostarch. A syrup, elixir or the like can be used wherein a sweetenedvehicle is employed. Sustained release compositions can be formulatedincluding those wherein the active component is protected withdifferentially degradable coatings, e.g., by microencapsulation,multiple coatings, etc.

In addition to treatment of human subjects, the therapeutic methods ofthe invention also will have significant veterinary applications, e.g.for treatment of livestock such as cattle, sheep, goats, cows, swine andthe like; poultry such as chickens, ducks, geese, turkeys and the like;horses; and pets such as dogs and cats. Also, the compounds of theinvention may be used to treat non-animal subjects, such as plants.

It will be appreciated that the actual preferred amounts of activecompounds used in a given therapy will vary according to the specificcompound being utilized, the particular compositions formulated, themode of application, the particular site of administration, etc. Optimaladministration rates for a given protocol of administration can bereadily ascertained by those skilled in the art using conventionaldosage determination tests conducted with regard to the foregoingguidelines.

In general, compounds of the invention for treatment can be administeredto a subject in dosages used in prior tetracycline therapies. See, forexample, the Physicians' Desk Reference. For example, a suitableeffective dose of one or more compounds of the invention will be in therange of from 0.01 to 100 milligrams per kilogram of body weight ofrecipient per day, preferably in the range of from 0.1 to 50 milligramsper kilogram body weight of recipient per day, more preferably in therange of 1 to 20 milligrams per kilogram body weight of recipient perday. The desired dose is suitably administered once daily, or severalsub-doses, e.g. 2 to 5 sub-doses, are administered at appropriateintervals through the day, or other appropriate schedule.

It will also be understood that normal, conventionally known precautionswill be taken regarding the administration of tetracyclines generally toensure their efficacy under normal use circumstances. Especially whenemployed for therapeutic treatment of humans and animals in vivo, thepractitioner should take all sensible precautions to avoidconventionally known contradictions and toxic effects. Thus, theconventionally recognized adverse reactions of gastrointestinal distressand inflammations, the renal toxicity, hypersensitivity reactions,changes in blood, and impairment of absorption through aluminum,calcium, and magnesium ions should be duly considered in theconventional manner.

Furthermore, the invention also pertains to the use of a tetracyclinecompound of formula I, for the preparation of a medicament. Themedicament may include a pharmaceutically acceptable carrier and thetetracycline compound is an effective amount, e.g., an effective amountto treat a tetracycline responsive state.

In yet another embodiment, the invention also pertains to the use of atetracycline compound of formula Ito treat a tetracycline responsivestate, e.g., in a subject, e.g., a mammal, e.g., a human.

Compounds of the invention may be made as described below, withmodifications to the procedure below within the skill of those ofordinary skill in the art.

Example 1 Synthesis of 7-, 8- and 9-Substituted Tetracycline Compounds7- and 9-Iodo Tetracycline Compounds

Five grams of a tetracycline compound was dissolved in 85 mL ofconcentrated sulfuric acid that was cooled to 0° C. (on ice).N-iodosuccinimide (NIS) was added to the reaction in 300 mg portionsevery 15 minutes. The reaction proceeded for 5 hours before beingremoved from the ice bath. The reaction mixture was then analyzed byHPLC and TLC, showed the product of D-ring iodotetracyclines. After thereaction was complete, the sulfuric acid was dripped slowly 1 L of icewater and extracted 7 times with 300 mL of n-butanol. The solvent wasremoved in vacuo to produce a mixture of three products. The 7-iodoregioisomer, 9-regioisomer and 7,9-diiodo tetracycline compoundderivatives were purified by preparative HPLC chromatography and bymethods known in the art.

9-NO₂ Doxycycline

1.0 g of doxycycline HCl was dissolved in concentrated H₂SO₄ (5 ml) andNaNO₃ (1.1 eq) was added over 1 minute. The reaction was stirred for 1hour and subsequently dripped into cold, rapidly stirred ether (500 ml).The precipitate was washed with ether and dried in vacuum to yield 9-NO₂doxycycline without further purification.

9-NH₂ Doxycycline

9-NO₂ doxycycline (1 g) was dissolved in methanol (50 ml) and pouredinto a Parr apparatus with 100 mg of 10% Pd/C. The reaction was chargedwith H₂ and shaken for 2 hours. The 9-amino doxycycline was separated bypreparative apic purification to produce 9-NH₂ doxycycline and 7-NH₂doxycycline in a 7:2 ratio.

9-N₂ Doxycycline

9-NH₂ doxycycline (1.5 g) was dissolved in 50 ml of 0.1N methanol HCland 2.2 ml of butyl nitrate was subsequently added. The reaction wasstirred for 1 hour, then the product was precipitated in 400 mL of dryether to produce the 9-diazonium salt of doxycycline

9-Azido Doxycycline

Sodium azide (0.126 g) was added to a warm 0.1 N methanol/HCl solutionof the 9-diazonium salt (1 g). The mixture was then stirred at roomtemperature for 4 hours and the product was obtained by precipitationinto diethyl ether. The solid was collected to yield 87% of the desiredproduct (9-azido doxycycline).

9-NH₂-8-Bromo Doxycycline

0.5 g of 9-azido doxycycline was added to 10 mL of HBr in acetic acid(30 wt %) and the reaction was stirred at room temperature for 1 hour.The product was obtained as a yellow powder in 56% yield by preparativeC18 HPLC.

8-Bromo Doxycycline

100 mg of 9-amino-8-bromo doxycycline was reacted with 1.1 g of butylnitrate in 0.1N HCl to produce the 9-diazonium salt of the 8-bromodoxycycline. The reaction was treated with phosphoric acid to yield theproduct as crude solid. Preparative HPLC produced the product as ayellow solid in 64% yield.

7-Phenyl Tetracycline

7-iodotetracycline (0.37 mmol), 30 mg Pd(PPh₃)₂Cl₂, 10 mg AsPh₃, and 6mg of CuI was dissolved in 25 ml of toluene with stirring. Phenyltri-n-butyltin (0.050 mL) was added and the solution was refluxed for 6hours under nitrogen. The solution was then cooled to room temperature,filtered, the solvent removed and the crude residue purified bypreparative thin layer chromatography.

Example 2 In vitro Minimum Inhibitory Concentration (MIC) Assay

The following assay is used to determine the efficacy of tetracyclinecompounds against common bacteria. 2 mg of each compound is dissolved in100 μl of DMSO. The solution is then added to cation-adjusted MuellerHinton broth (CAMHB), which results in a final compound concentration of200 μg per ml. The tetracycline compound solutions are diluted to 50 μLvolumes, with a test compound concentration of 0.098 μg/ml. Opticaldensity (OD) determinations are made from fresh log-phase broth culturesof the test strains. Dilutions are made to achieve a final cell densityof 1×10⁶ CFU/ml. At OD=1, cell densities for different genera should beapproximately:

E. coli 1 × 10⁹ CFU/ml S. aureus 5 × 10⁸ CFU/ml Enterococcus sp. 2.5 ×10⁹ CFU/ml  

50 μl of the cell suspensions are added to each well of microtiterplates. The final cell density should be approximately 5×10⁵ CFU/ml.These plates are incubated at 35° C. in an ambient air incubator forapproximately 18 hr. The plates are read with a microplate reader andare visually inspected when necessary. The MIC is defined as the lowestconcentration of the tetracycline compound that inhibits growth.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents are considered tobe within the scope of the present invention and are covered by thefollowing claims. The contents of all references, issued patents, andpublished patent applications cited throughout this application arehereby incorporated by reference. The appropriate components, processes,and methods of those patents, applications and other documents may beselected for the present invention and embodiments thereof.

1. A compound of formula (I):

or a pharmaceutically acceptable salt thereof, wherein: R⁴ and R^(4′)are each methyl; R⁵ is hydrogen or hydroxyl; to R⁶ and R^(6′) are eachindependently hydrogen, methyl, or hydroxyl; R⁷ is hydrogen, loweralkenyl, lower alkynyl, acyl, phenylalkynyl, dimethylamino or dioxenyl;R⁸ is hydrogen, phenyl, nitrophenyl, halo or lower alkynyl; and R⁹ ishydrogen, amino, acetamide or lower alkynyl; provided that at least oneof R⁷, R⁸, or R⁹ is not hydrogen.
 2. The compound of claim 1, wherein R⁵is hydroxyl, R⁶ is methyl, and R^(6′) is hydrogen.
 3. The compound ofclaim 2, wherein R⁷ is ethenyl, ethynyl, acyl, phenylethynyl ordioxenyl, and R⁸ and R⁹ are each hydrogen.
 4. The compound of claim 3,wherein said compound is 7-ethenyl doxycycline or 7-ethynyl doxycycline.5. The compound of claim 3, wherein said compound is 7-(1,4-dioxenyl)doxycycline, 7-acyl doxycycline or 7-acetyl doxycycline.
 6. The compoundof claim 3, wherein said compound is 7-phenylethynyl doxycycline.
 7. Thecompound of claim 2, wherein R⁸ is phenyl, bromo, or nitrophenyl; and R⁷and R⁹ are hydrogen.
 8. The compound of claim 7, wherein said compoundis 8-phenyl doxycycline, 8-bromo doxycycline or 8-(p-nitrophenyl)doxycycline.
 9. The compound of claim 2, wherein R⁸ is phenyl orethynyl, and R⁹ is amino.
 10. The compound of claim 9, wherein saidcompound is 8-ethynyl-9-amino doxycycline or 8-phenyl-9-aminodoxycycline.
 11. The compound of claim 2, wherein R⁹ is acetamide, andR⁷ and R⁸ are each hydrogen.
 12. The compound of claim 11, wherein saidcompound is 9-acetamide doxycycline.
 13. The compound of claim 1,wherein R⁵ is hydrogen, R⁶ is hydrogen, R^(6′) is hydrogen, R⁷ isdimethylamino, R⁸ is hydrogen and R⁹ is ethynyl.
 14. The compound ofclaim 13, wherein said compound is 9-ethynyl minocycline.
 15. Apharmaceutical composition comprising a therapeutically effective amountof a compound of claim 1 and a pharmaceutically acceptable carrier. 16.The pharmaceutical composition of claim 15, wherein said compound isselected from the group consisting of: 7-ethynyl doxycycline, 7-acyldoxycycline, 7-phenylethynyl doxycycline, 7-(1,4-dioxenyl) doxycycline,7-ethenyl doxycycline, 9-acetamide doxycycline, 8-phenyl-9-aminodoxycycline, 9-ethynyl minocycline, 8-bromo doxycycline, 8-phenyldoxycycline, 8-(p-nitrophenyl) doxycycline, 7-acetyl doxycycline and8-ethynyl-9-amino doxycycline.
 17. A method for treating a tetracyclineresponsive state in a mammal, comprising administering to said mammal acompound of claim
 1. 18. The method of claim 17, wherein said compoundis selected from the group consisting of: 7-ethynyl doxycycline, 7-acyldoxycycline, 7-phenylethynyl doxycycline, 7-(1,4-dioxenyl) doxycycline,7-ethenyl doxycycline, 9-acetamide doxycycline, 8-phenyl-9-aminodoxycycline, 9-ethynyl minocycline, 8-bromo doxycycline, 8-phenyldoxycycline, 8-(p-nitrophenyl) doxycycline, 7-acetyl doxycycline and8-ethynyl-9-amino doxycycline.
 19. The method of claim 17, wherein saidtetracycline responsive state is a bacterial infection.
 20. The methodof claim 19, wherein said bacterial infection is associated with E.coli, S. aureus or E. faecalis.